Bivalent binding molecules of 7 transmembrane G protein-coupled receptors

ABSTRACT

Described herein are methods for identifying and preparing bivalent binding molecules to 7 transmembrane G protein-coupled receptors. The methods disclosed herein are based on the SELEX method for generating high affinity nucleic acid ligands. SELEX is an acronym for Systematic Evolution of Ligands by EXponential enrichment. The methods of this invention combine two or more binding domains to two or more different epitopes of the same 7 transmembrane G protein-coupled receptor. These bivalent binding molecules are useful as therapeutic and diagnostic agents.

RELATEDNESS OF THE APPLICATION

[0001] The subject application is a divisional application of copendingU.S. Ser. No. 09/118,525, filed Jul. 17, 1998; which is acontinuation-in-part of U.S. Ser. No. 08/956,699, filed Oct. 23, 1997,now U.S. Pat. No. 6,083,696; which is a continuation of U.S. Ser. No.08/234,997, filed Apr. 28, 1994, now U.S. Pat. No. 5,683,867; each ofwhich are hereby incorporated in their entirety by reference.

FIELD OF THE INVENTION

[0002] Described herein are bivalent binding molecules that can activateor inhibit 7 transmembrane G protein-coupled receptors. Also describedherein are methods for identifying and preparing bivalent bindingmolecules to 7 transmembrane G protein-coupled receptors. The methodsdisclosed herein are based on the SELEX method for generating highaffinity nucleic acid ligands, also termed aptamers. SELEX is an acronymfor Systematic Evolution of Ligands by EXponential enrichment. Thebivalent binding molecules of this invention comprise two or morebinding domains to two or more different epitopes of the same 7transmembrane G protein-coupled receptor. In a preferred embodiment, atleast one binding domain is an aptamer. These bivalent binding moleculesare useful as therapeutic and diagnostic agents.

BACKGROUND OF THE INVENTION

[0003] The seven transmembrane receptors (also known as Gprotein-coupled receptors or 7TM G protein-coupled receptors) comprise asuperfamily of structurally related integral proteins. 7TM Gprotein-coupled receptors exhibit detectable amino acid sequencesimilarity and all appear to share a number of structural features (See,FIG. 1). These features include: an extracellular amino terminus (EAT);seven predominantly hydrophobic alpha-helical domains (of about 20-30amino acids) which are believed to span the cell membranes and arereferred to as transmembrane domains (TMD1-7); six loops which connectthe transmembrane domains (three extracellular loops (ELs) and threeintracellular loops (ILs)); and a cytoplasmic carboxy terminus (CCT).

[0004] Each 7TM G protein-coupled receptor is predicted to associatewith a particular heterotrimeric G protein (composed of α, β and γsubunits) at the intracellular surface of the plasma membrane. Uponbinding of an agonist to the receptor, a conformational change occurs inthe receptor, which enables interaction of the intracellular loops ofthe receptor with its associated intracellular, membrane-anchoredheterotrimeric G protein. This causes the alpha-subunit of the G proteinto exchange a bound GDP molecule for a GTP molecule and to dissociatefrom the β and γ subunits. The GTP-bound form of the alpha-subunit inturn stimulates specific intracellular signal-transducing enzymes andchannels.

[0005] It has been proposed that 7TM G protein-coupled receptors adopttwo major conformations: an active, G protein-coupled and thustransducing conformation and an inactive (non-transducing) conformation(Schwartz, T. W. et al., Cur. Pharmaceut. Design, 1:325-342 (1995)). Thebinding of an agonist or antagonist selectively stabilizes the activeand the inactive receptor conformations, respectively, as predicted bythe allosteric regulation of proteins as suggested by Monod, Wymann andChangeux (J. Mol. Biol., 263:7439-7442 (1965)). Agonists are thusextracellularly acting allosteric ligands that increase the signaltransduction rate at intracellular sites upon binding. Antagonists areextracellularly acting ligands that inhibit signal transduction uponbinding.

[0006] The 7TM G protein-coupled receptors are the largest family ofcell-surface receptors comprising several hundred distinct receptors,and over 100 receptors have been cloned. The transmembrane segments of7TM G protein-coupled receptor family members exhibit considerablehomology, whereas the extracellular connecting loops are less conserved,showing high homology only between closely related receptor subtypes.The 7TM G protein-coupled receptors can be grouped based on theirhomology levels and/or the nature of the ligands they recognize. Forexample, the interleukin-8 receptor, the angiotensin II receptor, thethrombin receptor, the endothelin receptors, the N-formyl peptidereceptor and the C5a receptor all bind peptide ligands and share 20-40%amino acid similarity.

[0007] The 7TM G protein-coupled receptors bind a wide variety ofligands of different molecular size ranging from small monoamines andother small molecules, to large neurotransmitters and peptide hormones.The family of 7TM G protein-coupled receptors also includes thereceptors for light (rhodopsin), for odors (olfactory receptors) and fortaste (gustatory receptors). Additionally, the conserved structure among7TM G protein-coupled receptors has allowed for the cloning of manynovel genes encoding 7TM G protein-coupled receptors whose naturalligand and function are yet to be elucidated. These receptors arereferred to as “orphan” receptors. Table 1 lists a number of 7TM Gprotein-coupled receptors which have been cloned and expressed.

[0008] Because of the involvement of 7TM G-protein-coupled receptors inthe regulation of many critically important biological functions anddisease conditions, many of these functions and conditions may beinfluenced or determined by the state of activation or inhibition (e.g.,blockade) of a 7TM G protein-coupled receptor. However, these receptorsare difficult to purify. The proteins can be removed from the membraneonly by the action of detergents, which denatures some proteins. Inaddition, most membrane proteins are not soluble in water. To date, fewnovel agonists or antagonists to these receptors have been identified.Common methods have involved generating antibodies to 7TM Gprotein-coupled receptors expressed in cells which have beenadministered to a host. Lerner et al. (PCT application No. WO 98/03632)have described peptide dimer agonists for 7TM G protein-coupledreceptors. These dimers were comprised of two known peptide agonists orantagonists (e.g., natural ligands) to different 7TM G protein-coupledreceptors.

[0009] It would be useful to be able to develop agonists and antagoniststo the specific binding portions of 7TM G protein-coupled receptors.Attempts to achieve expression of only the ligand binding portion of a7TM G protein-coupled receptor have been unreproducible or have resultedin inefficient and/or unpredictable levels of expression (Xie, U. B., etal, J. Biol. Chem. 265:21441-21420 (1990); Tsai-Morris, C. H., et al. J.Biol. Chem. 265:19385-19388 (1990)).

[0010] As suggested in Lerner et al., bivalent binding molecules canhave utility as therapeutics. More specifically, bivalent and bispecificantibodies have many practical applications, including inimmunodiagnosis and therapy. Bivalency can allow antibodies to bind tomultimeric antigens with great avidity; multivalency theoretically canincrease apparent binding affinity by several orders of magnitude(Crothers, D. M. et al., Immunochemistry 9: 341-351 (1972)).Bispecificity can allow the cross-linking of two antigens, for example,in recruiting cytotoxic T cells to mediate killing of a tumor cell.Specific examples of bivalent molecules capable of binding to adjacentepitopes include small bivalent antibodies composed of either antibodyfragments (F_(ab)) or single chain antibodies (F_(v)) (Pack, P. et al.,Biochemistry 31, 1579-1584 (1992); Holliger, P. et al., Proc. Natl.Acad. Sci. USA 90, 6444-6448 (1993); Mallender, W. D. et al., J. Biol.Chem., 269: 199-206 (1994)). Neri, D. et al. (J. Mol. Biol., 246:367-373(1995)) developed a bispecific antibody fragment, binding two antibodieswith a polypeptide chain, that recognizes adjacent and non-overlappingepitopes of lysozyme and is able to bind both epitopes simultaneously.

[0011] Bivalent peptides, such as receptor-adhesive modular proteins(“RAMPs”), have been used in an alternative approach to cell targeting.M. Engel et al., (Biochemistry 30: 3161-3169 (1991)) and C. A. Slate etal., (Int. J. Peptide Protein Res. 45: 290-298 (1995)) have designedlarge synthetic peptides, which contain two ligand sites separated by aspacer region and a dimerization domain.

[0012] The SELEX Process

[0013] A method for the in vitro evolution of nucleic acid moleculeswith highly specific binding to target molecules has been developed.This method, Systematic Evolution of Ligands by EXponential Enrichment,termed the SELEX process, is described in U.S. patent application Ser.No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by Exponential Enrichment,” now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands,” now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Nucleic Acid Ligands,”now U.S. Pat. No. 5,270,163 (see also WO 91/19813), each of which isherein specifically incorporated by reference. These applications,collectively referred to herein as the SELEX patent applications,describe a fundamentally novel method for making a nucleic acid ligandto any desired target molecule.

[0014] The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, preferably comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity nucleic acid ligands tothe target molecule.

[0015] The basic SELEX method has been modified to achieve a number ofspecific objectives. For example, U.S. patent application Ser. No.07/960,093, filed Oct. 14, 1992, entitled “Method for Selecting NucleicAcids on the Basis of Structure”, abandoned in favor of U.S. Ser. No.08/198,670, now U.S. Pat. No. 5,707,796, describes the use of SELEX inconjunction with gel electrophoresis to select nucleic acid moleculeswith specific structural characteristics, such as bent DNA. U.S. patentapplication Ser. No. 08/123,935, filed Sep. 17, 1993, entitled“Photoselection of Nucleic Acid Ligands”, abandoned in favor of U.S.Ser. No. 08/443,959, filed May 18, 1995, which was abandoned in favor ofU.S. Ser. No. 08/612,895, filed Sep. 16, 1994, now U.S. Pat. No.5,763,177, describes a SELEX-based method for selecting nucleic acidligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled“High-Affinity Nucleic Acid Ligands That Discriminate BetweenTheophylline and Caffeine”, now U.S. Pat. No. 5,580,737, describes amethod for identifying highly specific nucleic acid ligands able todiscriminate between closely related molecules, termed Counter-SELEX.U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993,entitled “Systematic Evolution of Ligands by EXponential Enrichment:Solution SELEX”, abandoned in favor of U.S. Ser. No. 08/461,069, filedJun. 5, 1995, now U.S. Pat. No. 5,567,588, and U.S. patent applicationSer. No. 08/792,075, filed Jan. 31, 1997, entitled “Flow Cell SELEX”,now U.S. Pat. No. 5,861,254, describe SELEX-based methods which achievehighly efficient partitioning between oligonucleotides having high andlow affinity for a target molecule. U.S. patent application Ser. No.07/964,624, filed Oct. 21, 1992, entitled “Nucleic Acid Ligands toHIV-RT and HIV-1 Rev”, now U.S. Pat. No. 5,496,938, describes methodsfor obtaining improved nucleic acid ligands after the SELEX process hasbeen performed. U.S. patent application Ser. No. 08/400,440, filed Mar.8, 1995, entitled “Systematic Evolution of Ligands by EXponentialEnrichment: Chemi-SELEX”, now U.S. Pat. No. 5,705,337, describes methodsfor covalently linking a ligand to its target.

[0016] The SELEX method encompasses the identification of high-affinitynucleic acid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. patent application Ser. No.08/117,991, filed Sep. 8, 1993, entitled “High Affinity Nucleic AcidLigands Containing Modified Nucleotides”, abandoned in favor of U.S.Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describesoligonucleotides containing nucleotide derivatives chemically modifiedat the 5- and 2′-positions of pyrimidines. U.S. patent application Ser.No. 08/134,028, now U.S. Pat. No. 5,580,737, supra, describes highlyspecific nucleic acid ligands containing one or more nucleotidesmodified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or 2′-O-methyl(2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22,1994, entitled “Novel Method of Preparation of Known and Novel 2′Modified Nucleosides by Intramolecular Nucleophilic Displacement”, nowU.S. Pat. No. 5,756,703, describes oligonucleotides containing various2′-modified pyrimidines.

[0017] The SELEX method encompasses combining selected oligonucleotideswith other selected oligonucleotides and non-oligonucleotide functionalunits as described in U.S. patent application Ser. No. 08/284,063, filedAug. 2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX”, now U.S. Pat. No. 5,637,459 and U.S. patentapplication Ser. No. 08/234,997, filed Apr. 28, 1994, entitled“Systematic Evolution of Ligands by Exponential Enrichment: BlendedSELEX”, now U.S. Pat. No. 5,683,867, respectively. The SELEX methodfurther encompasses combining selected nucleic acid ligands withlipophilic or Non-Immunogenic, High Molecular Weight compounds in adiagnostic or therapeutic complex as described in U.S. patentapplication Ser. No. 08/434,465, filed May 4, 1995, entitled “NucleicAcid Ligand Complexes”, now U.S. Pat. No. 6,011,020. VEGF Nucleic AcidLigands that are associated with a Lipophilic Compound, such as diacylglycerol or dialkyl glycerol, in a diagnostic or therapeutic complex aredescribed in U.S. patent application Ser. No. 08/739,109, filed Oct. 25,1996, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes”, now U.S. Pat. No. 5,859,228. VEGF Nucleic AcidLigands that are associated with a Lipophilic Compound, such as aglycerol lipid, or a Non-Immunogenic, High Molecular Weight Compound,such as polyalkylene glycol, are further described in U.S. patentapplication Ser. No. 08/897,351, filed Jul. 21, 1997, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”. VEGFNucleic Acid Ligands that are associated with a non-immunogenic, highmolecular weight compound or lipophilic compound are also furtherdescribed in PCT application Publication No. WO 98/18480, filed Oct. 17,1997, entitled “Vascular Endothelial Growth Factor (VEGF) Nucleic AcidLigand Complexes”. These applications allow the combination of the broadarray of shapes and other properties, and the efficient amplificationand replication properties, of oligonucleotides with the desirableproperties of other molecules. Each of the above described patentapplications which describe modifications of the basic SELEX procedureare specifically incorporated by reference herein in their entirety.

[0018] The identification of nucleic acid ligands to small, flexiblepeptides via the SELEX method has been explored. Small peptides haveflexible structures and usually exist in solution as an equilibrium ofmultiple conformers, and thus it was initially thought that bindingaffinities may be limited by the conformational entropy lost uponbinding a flexible peptide. However, the feasibility of identifyingnucleic acid ligands to small peptides in solution was demonstrated inU.S. Pat. No. 5,648,214, filed Sep. 9, 1994, entitled “High-AffinityOligonucleotide Ligands to the Tachykinin Substance P”, which isincorporated herein by reference. In this patent, high affinity RNAnucleic acid ligands to substance P, an 11 amino acid peptide wereidentified.

[0019] Xu and Ellington (Proc. Natl. Acad. Sci. USA, 93:7475-7480(1996)) employed the human immunodeficiency virus type 1 (HIV-1) Rev tofurther explore how peptide and protein epitopes are recognized bynucleic acid ligands. In this study, RNA nucleic acid ligands wereselected to bind to the isolated Rev₃₄₋₅₀ peptide. It was observed thatRNA nucleic acid ligands could not only recognize the sequence of thispeptide, but that these nucleic acid ligands could also bind thecorresponding native epitope on the Rev protein, albeit with loweraffinity.

[0020] The present invention provides bivalent binding moleculescomprising two or more binding domains which bind simultaneously to twoor more epitopes of the same 7TM G protein-coupled receptor and thusincrease the binding affinity relative to the binding of a singlebinding domain. The binding domains are identified using syntheticpeptides corresponding to all or a portion of the extracellular bindingdomains and therefore purified and isolated receptor proteins are notrequired.

BRIEF SUMMARY OF THE INVENTION

[0021] The present invention describes bivalent binding molecules havingbinding affinity for two or more epitopes of the same 7TM Gprotein-coupled receptor and methods for generating and producing suchbivalent binding molecules. The bivalent binding molecules describedherein may be agonists, antagonists or superagonists. In one embodimentthe bivalent binding molecules of the invention comprise a first bindingdomain coupled to a second binding domain, the first and second bindingdomains being ligands to a first and second epitopes. In preferredembodiments, the first and second epitopes are located on differentextracellular loops of the same 7TM G protein-coupled receptor. In apreferred embodiment, at least one binding domain of the bivalentbinding molecule is an aptamer. In one embodiment, the first and secondbinding domains are coupled via a linker.

[0022] In a preferred embodiment, both the first and second bindingdomains are aptamers to first and second epitopes of two differentextracellular loops. In this embodiment, the aptamers are coupled toeach other at either their 5′ or 3′ ends. In a preferred embodiment, theaptamers are SELEX-derived aptamers.

[0023] In another embodiment, the first binding domain is an aptamer toa first epitope of a 7TM G protein-coupled receptor, and the secondbinding domain is a non-aptamer binding domain that binds to a secondepitope of the same 7TM G protein-coupled receptor.

[0024] In one embodiment of the method of this invention, a method foridentifying bivalent binding molecules to 7TM G protein-coupledreceptors is described, wherein the bivalent binding molecules comprisetwo binding domains, each of which is an aptamer. In this method, thebivalent binding molecules are identified generally by applying theChimeric SELEX methods described in U.S. Pat. No. 5,637,459, filed Aug.2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX”, which is incorporated herein by reference.

[0025] In another embodiment of the method of this invention, a methodfor identifying bivalent binding molecules to 7TM G protein-coupledreceptors is described, wherein one binding domain of the bivalentmolecule is an aptamer and the other is a non-aptamer binding domain. Inthis method, the bivalent compounds are identified generally by applyingthe Blended SELEX methods described in U.S. Pat. No. 5,683,867, filedApr. 28, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Blended SELEX”, which is incorporated herein by reference.Suitable non-aptamer binding domains include all or a portion of anatural ligand to the 7TM G protein-coupled receptor.

[0026] In another embodiment of the method of this invention, a methodfor identifying bivalent binding molecules to 7TM G protein-coupledreceptors is described, wherein both binding domains are unnaturalL-aptamers (mirror images of the natural D-aptamers). In otherembodiments, the first binding domain is an aptamer of non-naturalhandedness to a first epitope and the second binding domain is either anaptamer of natural handedness or a non-aptamer binding domain to asecond epitope.

[0027] This invention further includes the bivalent binding molecules to7TM G protein-coupled receptors generated by the methods of thisinvention. These bivalent binding molecules may contain one or moremodified nucleotides such as nucleotides modified at the 2′- and/or 5and/or 8 positions. Such modifications include but are not limited tonucleotides containing 2′-amino (2′-NH₂), 2′-fluoro (2′-F) and2′-O-methyl (2′-O-Me) groups.

[0028] With certain 7TM G protein-coupled receptors, it may not benecessary for a binding ligand to be bivalent. Therefore this inventionfurther includes aptamers to 7TM G protein-coupled receptors identifiedby the SELEX process which can act as agonists or antagonists of thereceptor.

[0029] This invention further provides a method for treating diseases orconditions mediated by 7TM G protein-coupled receptors.

BRIEF DESCRIPTION OF THE FIGURES

[0030]FIG. 1 illustrates the structure of a representative 7TM Gprotein-coupled receptor. Symbols used are EAT (extracellularamino-terminus), which is encircled by the dotted line; CM (cellularmembrane); 1-3IL (first through third intracellular loops); 1-3EL (firstthrough third extracellular loops); TMD 1-7 (first through seventhtransmembrane domains); and CCT (cytoplasmic carboxy-terminus).

DETAILED DESCRIPTION OF THE INVENTION

[0031] This invention describes bivalent binding molecules havingbinding affinity for two or more epitopes of the same 7TM Gprotein-coupled receptor. In one embodiment the bivalent bindingmolecules comprise a first binding domain to a first epitope on a firstextracellular loop of a 7TM G protein-coupled receptor coupled to asecond binding domain to a second epitope on a second extracellular loopof the same 7TM G protein-coupled receptor. In a preferred embodiment atleast one binding domain is an aptamer. Also included in this inventionare methods for identifying and producing aptamers and bivalent bindingmolecules to 7TM G protein-coupled receptors. The methods describedherein are based on the SELEX method.

[0032] 1. Definitions

[0033] A “bivalent binding molecule” is a compound comprising two ormore binding domains having binding affinity to two or more epitopes ofthe same 7 transmembrane G protein-coupled receptor. In a preferredembodiment at least one of the binding domains is an aptamer. While themajority of the embodiments of bivalent binding molecules disclosedherein are described in terms of having two binding domains, it is to beunderstood that this invention also includes binding molecules havingthree binding domains to three different epitopes of the same 7transmembrane G protein-coupled receptor. Furthermore it is to beunderstood that these embodiments also include binding molecules havingfour binding domains to 4 different epitopes on the same 7TM Gprotein-coupled receptor. The epitopes for which the binding domains areselected may be on the same or different extracellular domains and neednot be on adjacent extracellular domains. In a preferred embodiment, allof the binding domains are aptamers. The binding domains may be coupledto each other via a linker. In another embodiment, one of the aptamerbinding domains may be replaced by a non-aptamer binding domain thatbinds to an epitope of the same 7 transmembrane G protein-coupledreceptor. The bivalent binding molecules may comprise binding domainswhich are all agonists, all antagonists or the bivalent molecule maycomprise a mixture of agonist and antagonist binding domains.

[0034] A “binding domain” is an entity that binds to all or a portion ofan extracellular domain of a 7TM G protein-coupled receptor. The bindingdomain may be a nucleic acid ligand (aptamer), an antibody or antibodyfragment, or may be all or a portion of a naturally occurring ligand ofthe 7TM G protein-coupled receptor.

[0035] An “epitope” is a protein domain of a receptor. For the purposesof this invention an epitope may be an entire extracellular portion or asubportion of the extracellular amino terminus or the first throughthird extracellular loops of a 7TM G protein-coupled receptor as shownin FIG. 1. The epitope may be a synthetic peptide or a recombinantlyexpressed protein or protein fragment. The 7TM G protein coupledreceptor may be expressed in a cell.

[0036] A “7 transmembrane G protein-coupled receptor” is a large proteinsuch as that shown in FIG. 1 that transmits a signal from one side of acell membrane to the other side of the membrane upon binding of anagonist. Members of the superfamily of 7 transmembrane G protein-coupledreceptors include those which are listed in Table 1. Examples ofbiological functions mediated by 7TM G protein-coupled receptorsinclude, but are not limited to, hormone action, neurotransmission,chemotaxis, perception of light, smell and taste, and regulation ofblood pressure, heart rate and blood clotting.

[0037] A “linker” is a molecular entity that connects two or moremolecular entities, and can allow spatial separation of the molecularentities in a manner that preserves the functional properties of one ormore of the molecular entities. A linker can also be known as a spacer.Suitable linkers include, but are not limited to, polymers includingpolyethylene glycol, polypropylene glycol, polyvinyl alcohol,hydrocarbons, polyacrylates and amino-, hydroxy-, thio orcarboxy-functionalized silicones; proteins; peptides; polynucleotides;saccharides including monosaccharides, oligosaccharides, cyclodextrinsand dextran; other biocompatible moieties; and combinations thereof. Alinker may also be a liposome.

[0038] An “aptamer” or “nucleic acid ligand” as used herein is anon-naturally occurring nucleic acid having a desirable action on atarget. A desirable action includes, but is not limited to, binding ofthe target, catalytically changing the target, reacting with the targetin a way which modifies/alters the target or the functional activity ofthe target, covalently attaching to the target as in a suicideinhibitor, and facilitating the reaction between the target and anothermolecule. In the preferred embodiment, the desirable action is specificbinding to a target molecule, such target molecule being a threedimensional chemical structure other than a polynucleotide that binds tothe nucleic acid ligand through a mechanism which predominantly dependson Watson/Crick base pairing or triple helix binding, wherein thenucleic acid ligand is not a nucleic acid having the known physiologicalfunction of being bound by the target molecule. For the purposes of thisinvention, an aptamer is a nucleic acid ligand having specific bindingaffinity for an epitope on an extracellular membrane of a 7TM Gprotein-coupled receptor.

[0039] “Nucleic acid” means either DNA, RNA, single-stranded ordouble-stranded and any chemical modifications thereof. Modificationsinclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability, hydrogenbonding, electrostatic interaction, and fluxionality to the nucleic acidligand bases or to the nucleic acid ligand as a whole. Suchmodifications include, but are not limited to, 2′-position sugarmodifications, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at exocyclic amines, substitution of4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbonemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

[0040] “Target” as used herein means any compound or molecule ofinterest for which an aptamer is desired. A target can be a protein,peptide, carbohydrate, polysaccharide, glycoprotein, hormone, receptor,antigen, antibody, virus, substrate, metabolite, transition stateanalog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell,tissue, etc., without limitation. In this application, the target is apeptide target comprising all or a portion of an epitope of a 7TM Gprotein-coupled receptor. A target may also be an enantiomer of anaturally occurring epitope.

[0041] An “agonist” is a compound that binds a receptor specific forthat compound and elicits a response, i.e., activates the receptor.Thus, the binding of an agonist to its receptor elicits a biologicalresponse mediated by the receptor. For the purposes of this invention,the binding of an agonist to a 7TM G protein-coupled receptor isbelieved to cause a conformational change that enables interaction ofthe intracellular loops of the receptor with its intracellular Gprotein.

[0042] An “antagonist” is a compound that binds to a receptor such thatthe compound interferes with the ability of an agonist of that receptorto evoke a response. Thus, the binding of an antagonist to the receptordoes not elicit a biological response mediated by the receptor. For thepurposes of this invention, binding of an antagonist to a 7transmembrane G protein-coupled receptor is believed to inhibit signaltransduction by causing a conformation that does not allow interactionof the intracellular loops of the receptor with its intracellular Gprotein.

[0043] A “superagonist” is a compound that binds to a receptor in amanner such that it traps a natural ligand in its binding site in thereceptor, thereby permanently activating that receptor.

[0044] “Lipid constructs”, for purposes of this invention, arestructures containing lipids, phospholipids, or derivatives thereofcomprising a variety of different structural arrangements which lipidsare known to adopt in aqueous suspension. These structures include, butare not limited to, lipid bilayer vesicles, micelles, liposomes,emulsions, lipid ribbons or sheets, and may be complexed with a varietyof drugs and components which are known to be pharmaceuticallyacceptable. In the preferred embodiment, the lipid construct is aliposome. The preferred liposome is unilamellar and has a relative sizeless than 200 nm. Common additional components in lipid constructsinclude cholesterol and alpha-tocopherol, among others.

[0045] “Lipid bilayer vesicles” are closed, fluid-filled microscopicspheres which are formed principally from individual molecules havingpolar (hydrophilic) and non-polar (lipophilic) portions. The hydrophilicportions may comprise phosphato, glycerylphosphato, carboxy, sulfato,amino, hydroxy, choline and other polar groups. Examples of non-polargroups are saturated or unsaturated hydrocarbons such as alkyl, alkenylor other lipid groups. Sterols (e.g., cholesterol) and otherpharmaceutically acceptable components (including anti-oxidants likealpha-tocopherol) may also be included to improve vesicle stability orconfer other desirable characteristics.

[0046] “Liposomes” are a subset of lipid bilayer vesicles and arecomprised principally of phospholipid molecules which contain twohydrophobic tails consisting of long fatty acid chains. Upon exposure towater, these molecules spontaneously align to form a bilayer membranewith the lipophilic ends of the molecules in each layer associated inthe center of the membrane and the opposing polar ends forming therespective inner and outer surface of the bilayer membrane. Thus, eachside of the membrane presents a hydrophilic surface while the interiorof the membrane comprises a lipophilic medium. These membranes whenformed are generally arranged in a system of concentric closed membranesseparated by interlamellar aqueous phases, in a manner not dissimilar tothe layers of an onion, around an internal aqueous space. Thesemultilamellar vesicles (MLV) can be converted into unilamellar vesicles(UV) with the application of a shearing force.

[0047] 2. The SELEX Method

[0048] The SELEX method is described in U.S. patent application Ser. No.07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution ofLigands by EXponential Enrichment”, now abandoned; U.S. patentapplication Ser. No. 07/714,131, filed Jun. 10, 1991, entitled “NucleicAcid Ligands”, now U.S. Pat. No. 5,475,096; U.S. patent application Ser.No. 07/931,473, filed Aug. 17, 1992, entitled “Nucleic Acid Ligands”,now U.S. Pat. No. 5,270,163, (see also WO 91/19813). These applications,each specifically incorporated herein by reference, are collectivelycalled the SELEX patent applications.

[0049] In its most basic form, the SELEX process may be defined by thefollowing series of steps:

[0050] 1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100 percent).

[0051] 2) The candidate mixture is contacted with the selected targetunder conditions favorable for binding between the target and members ofthe candidate mixture. Under these circumstances, the interactionbetween the target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target pairs between the target andthose nucleic acids having the strongest affinity for the target.

[0052] 3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5-50%) areretained during partitioning.

[0053] 4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

[0054] 5) By repeating the partitioning and amplifying steps above, thenewly formed candidate mixture contains fewer and fewer weakly bindingsequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids from theoriginal candidate mixture having the highest affinity to the targetmolecule.

[0055] The SELEX patent applications describe and elaborate on thisprocess in great detail. Included are targets that can be used in theprocess; methods for partitioning nucleic acids within a candidatemixture; and methods for amplifying partitioned nucleic acids togenerate an enriched candidate mixture. The SELEX patent applicationsalso describe ligands obtained to a number of target species, includingboth protein targets where the protein is and is not a nucleic acidbinding protein. Each of the above described patent applications whichdescribe modifications of the basic SELEX procedure are specificallyincorporated by reference herein in their entirety.

[0056] The present invention provides methods for generating bivalentbinding molecules and the bivalent binding molecules so produced. Abivalent molecule is defined as a molecule having two or more bindingdomains having affinity for two or more epitopes of the same 7TM Gprotein-coupled receptor. In one embodiment, the bivalent bindingmolecule comprises a first and second binding domain having affinity fora first and second epitope, respectively, of the same 7TM Gprotein-coupled receptor.

[0057] While not wishing to be bound by any theory, it is believed thatin embodiments wherein the bivalent binding molecules are agonists, thefirst and second binding domains bind to their respective epitopes onthe extracellular loops of the 7TM G protein-coupled receptor and causea conformation change to occur in the receptor which enables interactionof the intracellular loops of the receptor with its intracellular Gprotein.

[0058] While not wishing to be bound by any theory, it is believed thatin embodiments wherein the bivalent binding molecules are antagonists,the first and second binding domains bind to their respective epitopeson the extracellular loops of the 7TM G protein-coupled receptor andprevent the correct conformational change from occurring in the receptorwhich ensures that the intracellular loops of the receptor cannotinteract with its intracellular G protein.

[0059] Further, while not wishing to be bound by any theory, it isbelieved that in embodiments wherein the bivalent binding molecules aresuperagonists, the first and second binding domains bind to theirrespective epitopes on the extracellular loops of the same receptor in amanner which traps a natural agonist in its binding site in the samereceptor. As a result the natural agonist “locks” the intracellularloops in a conformation in which they “permanently” interact with the Gprotein of that receptor, and thus the receptor is in a continual stateof activation.

[0060] 3. Methods of Selecting and Synthesizing Epitopes

[0061] In a preferred embodiment, the binding domains of the bivalentcompounds described herein are binding domains to epitopes on theextracellular loops of a 7TM G protein-coupled receptor.

[0062] In one embodiment, both of the binding domains are aptamersidentified by the SELEX process, wherein the targets used to identifythe aptamers are epitopes, or protein domains of the 7TM Gprotein-coupled receptor. In order to identify and define a desiredtarget (i.e., the epitope), the protein corresponding to the receptor(preferably in an extracellular loop) of a 7TM G protein-coupledreceptor of interest must be defined. There are a number of methodsknown in the art for determining the various protein domains. Theseinclude hydrophobicity analysis to identify the hydrophobic andhydrophilic segments of the 7TM G protein-coupled receptor and thereforedetermine those parts of the receptor that comprise the extracellularloops (Heijne, G., J. Mol. Biol. 225:487-494 (1992); (Kyte, J., andDoolittle, R. F. (1982) J. Mol. Biol., 157:105-132)). The secondarystructure can also be analyzed to identify alpha-helix structures, whichmake up the hydrophobic regions of the 7TM G protein-coupled receptors.Computer-assisted structural assessment tools that can be used toexamine the hydrophobic and hydrophilic segments, the flexibility(Karplus, P. A. and Shultz, G. E. Naturwissenschaften, 72:212-213), andsecondary structure (Chou, P. Y. and Fasman, G. D. (1976) 47:251-276) oftransmembrane domains are also well known in the art (see, for example,Krystek et al. (1985) Endocrinology (Baltimore) 117:1125-1131).Correlation of flexibility plots with homology plots and surfaceprofiles may also be helpful in identifying specific regions of proteinstructure amongst members of the 7TM G protein-coupled receptor.

[0063] Once the protein domains of interest are identified, peptides maybe prepared consisting of amino acids corresponding to a desired proteindomain of the 7TM G protein-coupled receptor of interest. The peptideprepared may be the entire portion of the identified epitope or may be aportion of the epitope. Typically, the peptide comprises between 9 and100 consecutive amino acids, and preferably between 20 and 50consecutive amino acids corresponding to the protein domain.

[0064] The peptide may be prepared by synthetic methods or may berecombinantly expressed. Methods for peptide synthesis are known in theart and are described in Solid Phase Peptide Synthesis, (1984), byStewart and Young for synthesis by the solid-phase method of Merrifieldand in Houben Weyl Methoden der Otganischen Chemie, (1974), Vol. 16,parts I & II for solution-phase synthesis. Methods for solution phasesynthesis of peptides are also described in U.S. patent application Ser.No. 08/780,517, filed Jan. 8, 1997, entitled “Method for Solution PhaseSynthesis of Oligonucleotide and Peptides”, now U.S. Pat. No. 5,874,532,the contents of which are incorporated herein by reference. Methods ofrecombinantly expressing peptides are well known to those of skill inthe art.

[0065] 4. Methods of Generating Bivalent Binding Molecules

[0066] A. Generating Bivalent Binding Molecules by the Traditional SELEXMethod

[0067] In one embodiment, the invention includes bivalent bindingmolecules to 7TM G protein-coupled receptors comprising a first andsecond binding domain to a first and second epitope of the same 7TM Gprotein-coupled receptor, wherein the first and second binding domainsare aptamers identified generally according to the method known as theSELEX process. As stated earlier, the SELEX technology is described indetail in the SELEX patent applications which are incorporated herein byreference. In this embodiment, the first and second epitopes areidentified, and an aptamer to each epitope is identified by the SELEXmethod as follows.

[0068] An epitope of a 7TM G protein-coupled receptor is identified, andthe peptide target for aptamer selection (e.g., all or a portion of thepeptide sequence of the epitope) is synthesized by methods known in theart. A candidate mixture of random nucleic acids is prepared andcontacted with the peptide target. In general, any protocol which willallow selection of nucleic acids based on their ability to bindspecifically to the peptide target can be employed. For example, afilter binding selection, as described in U.S. Pat. No. 5,475,096, maybe employed in which a the candidate mixture is incubated with thepeptide target under conditions which will allow nucleic acid/peptidetarget binding pairs to form. The nucleic acid/peptide target bindingpairs are isolated by filtering the mixture through a nitrocellulosefilter and washing with an appropriate buffer to remove unbound nucleicacids.

[0069] In a preferred embodiment, the aptamers are identified bycovalently or noncovalently attaching the peptide target to a column(e.g., an affinity column) or other support matrix as described in U.S.Pat. No. 5,475,096. Any standard coupling reagent or procedure may beutilized, depending on the nature of the support. For example, covalentbinding may include the formation of disulfide, ester or amide linkages.Noncovalent linkages include antibody-antigen interactions or proteinsugar interactions. Other linking chemistries are also available. Forexample, disulfide-derivatized biotin (Pierce) may be linked to thepeptide by coupling through an amine or other functional group. Theresulting peptide-biotin complex could then be used with anavidin-derivatized support. Linking chemistries will be selected on thebasis of the conditions or reagents necessary for maintaining thestructure of the peptide and/or chemical groups on the peptide availablefor linking to the support matrix. The candidate mixture is added to andincubated with the support to allow nucleic acids having affinity to thepeptide to bind to the peptide. The nucleic acid/peptide target bindingpairs are separated from unbound nucleic acids by removing unboundnucleic acids from the support, for example, by washing the support withan appropriate buffer which will remove unbound nucleic acids but willnot remove the nucleic acid/peptide target binding pairs.

[0070] Following the identification of the aptamers for first and secondepitopes, the aptamers may be synthesized using standard methods knownin the art for synthesizing nucleic acids and then coupled, optionallyvia a linker. The aptamers may be coupled in any of the followingmanners: Aptamer1 Aptamer2 3′------------5′-L-5′------------3′3′------------5′-L-3′------------5′ 5′------------3′-L-5′------------3′5′------------3′-L-3′------------5′

[0071] wherein L represents an optional linker. Methods for couplingaptamers without a linker are known in the art. One method involvessolution or solid-phase synthesis of an oligonucleotide sequencecorresponding to the sequential combined sequences of both aptamers. Thelinker may be incorporated using any standard solid phaseoligonucleotide synthesis technique known in the art.

[0072] After the bivalent molecule is identified, the binding affinitymay be measured by methods known in the art. The binding affinity of thebivalent molecule may be tested on the isolated 7TM G protein-coupledreceptor or may be tested using a cell line which expresses the protein.

[0073] It may be desirable in the process of identifying bivalentbinding molecules to remove members of the bivalent molecule librarywhich bind to a second substance. This may be achieved by utilizing amethod known as Counter-SELEX, which is described in U.S. Pat. No.5,580,737, filed Oct. 7, 1993, entitled “High-Affinity Nucleic AcidLigands That Discriminate Between Theophylline and Caffeine”, and inU.S. Pat. No. 5,750,342, filed May 3, 1995, entitled “Nucleic AcidLigands of Tissue Target”. These patents are incorporated herein byreference in their entirety. In one embodiment, a positive selectionstep is first performed in which the bivalent molecule library is firstincubated with cells which express the 7TM G protein-coupled receptor ofinterest. Those skilled in the field of molecular biology willunderstand that any of a wide variety of expression systems may be usedto provide the recombinant receptor protein. The precise host cell usedis not critical to the invention. Cells to which the higher affinitybivalent binding molecules are bound are isolated and the bivalentbinding molecules are removed to provide an enriched pool of bivalentbinding molecules. In a second, negative selection step, the enrichedpool of bivalent binding molecules is then incubated with a second setof cells which are identical to the above cells but which do not expressthe 7TM G protein-coupled receptor. Those bivalent binding moleculeswhich bind to the second set of cells are discarded, and the remainingbivalent binding molecules are now enriched for bivalent bindingmolecules with high specificity for the 7TM G protein-coupled receptor.

[0074] In a second embodiment, the negative selection step may beperformed first with a cell line that does not express the 7TM Gprotein-coupled receptor of interest, followed by a positive selectionstep with a cell line that does express the 7TM G protein-coupledreceptor.

[0075] B. Generating Bivalent Binding Molecules by the Chimeric SELEXMethod

[0076] In one embodiment, the bivalent binding molecules of theinvention are generated by the Chimeric SELEX method. In thisembodiment, the binding domains comprise first and second aptamers tofirst and second epitopes, respectively, on the same 7TM Gprotein-coupled receptor. The Chimeric SELEX method combines two or moreSELEX-evolved nucleic acid ligands and/or nucleic acid libraries (alsoreferred to as parent libraries) into a single chimeric nucleic acidmolecule containing the functions of both parent libraries and/ornucleic acid ligands. The chimeric nucleic acid libraries generated mayserve as candidate mixtures for further evolution through the SELEXprocedure, or they may simply be partitioned for identification ofchimeric nucleic acid molecules having the desired characteristics. TheChimeric SELEX method is described in U.S. Pat. No. 5,637,459, filedAug. 2, 1994, entitled “Systematic Evolution of Ligands by ExponentialEnrichment: Chimeric SELEX”, the contents of which are incorporatedherein by reference.

[0077] A “chimeric nucleic acid library” or “chimeric aptamer library”is defined as a collection of molecules, each comprising two aptamerscoupled to each other. Each aptamer is derived from a separate parentaptamer library. In the present invention, the parent aptamer librariesare preferably comprised of nucleic acids generated following at leastone round of the SELEX process and are enriched in aptamers havingbinding affinity for two different epitopes of the same 7TM Gprotein-coupled receptor. The chimeric aptamer libraries of the presentinvention are also described as bivalent molecule libraries.

[0078] In one embodiment of this invention, a method for generatingbivalent binding molecules to 7TM G protein-coupled receptors isdescribed, which involves first generating a bivalent molecule libraryby a method comprising:

[0079] (a) generating a first library of aptamers selected through theSELEX procedure for a first epitope of a 7TM G protein-coupled receptor,wherein the aptamers have a 3′ fixed sequence, and producing thedouble-stranded form of the first library of aptamers;

[0080] (b) generating a second library of aptamers selected through theSELEX procedure for a second epitope of the same 7TM G protein-coupledreceptor, wherein the aptamers have a 5′ fixed sequence identical to the3′ fixed sequence of the nucleic acids of the first library, andproducing the double-stranded form of the second library of aptamers;

[0081] (c) mixing the first and second libraries under conditions whichpromote interlibrary annealing;

[0082] (d) generating bivalent binding molecules by enzymaticallyextending the recessed 3′ ends while copying the 5′ extensions of eachannealed pair; and

[0083] (e) amplifying the bivalent binding molecules to generatedouble-stranded DNA.

[0084] In embodiments of the invention where a single-stranded RNAbivalent molecule library is desired, the method further comprises thestep:

[0085] (f) transcription of the double-stranded DNA to yield asingle-stranded RNA bivalent molecule library.

[0086] In embodiments of the invention where a single-stranded DNAlibrary is desired, the method further comprises the step:

[0087] (f) separating the strands of the double-stranded DNA to yield asingle-stranded DNA bivalent molecule library.

[0088] The bivalent molecule libraries are then amplified, contactedwith the 7TM G protein-coupled receptor, and cycles of selection andamplification are performed to identify a high affinity bivalentmolecule to the 7TM G protein-coupled receptor.

[0089] In a preferred embodiment, the bivalent molecule library isenriched for higher affinity aptamers for the first and second epitopesprior to selection with the 7TM G protein-coupled receptor by the stepscomprising:

[0090] (g) contacting the bivalent molecule library with the firstepitope, wherein bivalent binding molecules having increased affinity tothe first epitope may be partitioned from the bivalent molecule library;

[0091] (h) partitioning the bivalent binding molecules having increasedaffinity to the first epitope from the remainder of the bivalentmolecule library;

[0092] (i) contacting the bivalent binding molecules having increasedaffinity to the first epitope with the second epitope, wherein bivalentbinding molecules having increased affinity to the first and secondepitope may be partitioned from the remainder of the bivalent bindingmolecules having increased affinity only for the first epitope, therebygenerating a bivalent molecule library enriched for bivalent bindingmolecules having increased affinity for the first and second epitopes.This enriched bivalent molecule library is then screened for memberswhich have high affinity for the 7TM G protein-coupled receptor.

[0093] In a preferred embodiment, bivalent binding molecules areselected for the 7TM G protein-coupled receptor which is expressed in acell. In one embodiment, a negative selection process is employed asdescribed above.

[0094] In another embodiment, the bivalent molecule library is generatedby placing a monophosphate at the 5′-end of only one of the two parentbivalent molecule libraries and members of the two parent libraries arejoined by enzymatic ligation. The 5′-monophosphate may be generated in anumber of ways, including by restriction digestion, kinasing, or bypriming transcription with a nucleotide monophosphate.

[0095] In another embodiment, the first and second libraries are coupledvia a linker.

[0096] C. Generating Bivalent Binding Molecules by the Blended SELEXMethod

[0097] In another embodiment, bivalent binding molecules of theinvention comprise an aptamer coupled to a non-aptamer binding domain.In this embodiment, the bivalent binding molecules of the invention aregenerated by the Blended SELEX method. The Blended SELEX method is amethod for combining nucleic acids with other functional units forgeneration of high affinity ligands. Blended SELEX is described in U.S.Pat. No. 5,683,867, filed Apr. 28, 1994, entitled “Systematic Evolutionof Ligands by Exponential Enrichment: Blended SELEX”, the contents ofwhich are incorporated herein by reference.

[0098] In one embodiment of the invention, bivalent binding molecules toa 7TM G protein-coupled receptor are prepared by performing the BlendedSELEX method utilizing a candidate mixture of nucleic acids wherein eachnucleic acid sequence of the candidate mixture is coupled to anon-aptamer binding domain known to bind to a second epitope of thereceptor to form a blended candidate mixture of bivalent bindingmolecules. The non-aptamer binding domain may be coupled at either the5′ or 3′ end of the nucleic acid sequence. Methods of couplingnon-aptamer binding domains to nucleic acid sequences are well withinthe skill of those ordinarily practicing in the art. The blendedcandidate mixture is contacted with the 7TM G protein-coupled receptorunder conditions appropriate for binding of members of the blendedcandidate mixture to the receptor. Those members of the candidatemixture which bind to the receptor are partitioned from the candidatemixture, amplified, and the bivalent binding molecules having highbinding affinity to the receptor are identified.

[0099] In another embodiment, bivalent binding molecules to a 7TM Gprotein-coupled receptor are prepared by performing the traditionalSELEX method using an candidate mixture of nucleic acids to identify anaptamer to a first epitope of the 7TM G protein-coupled receptor. Theaptamer is then covalently coupled to a non-aptamer binding domain of asecond epitope of the same 7TM G protein-coupled receptor to provide abivalent molecule. Useful non-aptamer binding domains may be all or aportion of a natural ligand to the receptor and include, but are notlimited to, antibodies, proteins (including peptides and polypeptides),or their derivatives, including, but limited to, hormones, antigens,synthetic or naturally occurring drugs, and the like; opiates; dopamine;serotonin; catecholamines; thrombin; acetylcholine; prostaglandins;small molecules such as fragrances; pheromones; adenosine; simple sugarssuch as sucrose, glucose, lactose and galactose; and any other moietiesthat recognize and have affinity towards an epitope of a 7TM Gprotein-coupled receptor.

[0100] D. Generating Bivalent Binding Molecules by Mirror-SymmetricalSelection

[0101] In another embodiment of the method of this invention, a methodfor identifying bivalent binding molecules to 7TM G protein-coupledreceptors is described, wherein the bivalent binding molecules comprisetwo binding domains, wherein one or both binding domains is an unnaturalL-nucleic acid ligand (a mirror image of the natural D-nucleic acidligand). In this method, one or both of the ligand components areidentified by the method described in PCT patent application No. WO96/34879, entitled “Identification of Enantiomeric Ligands” (Schumacheret al.), and in PCT application No. WO 98/08856, entitled“Mirror-Symmetrical Selection and Evolution of Nucleic Acids” (Fürste,et al.) which describes a method of identifying nucleic acid ligandswhich are of the opposite chirality from that which occurs in nature(DNA occurs in nature as a D isomer).

[0102] In this embodiment, the first step involves synthesizing anenantiomer of a peptide sequence corresponding to the amino acidsequence of the first and/or second epitope(s) of interest (e.g., a Damino acid peptide enantiomer of an L amino acid peptide). The peptideis contacted with a candidate mixture of nucleic acids of naturalhandedness (i.e., D-DNA or D-RNA) under conditions appropriate forbinding. The nucleic acids with high binding affinity for the D aminoacid peptide are isolated and their sequences are identified. Nucleicacids of non-natural handedness (i.e., L-DNA or L-RNA), which are mirrorimages of the high affinity D-DNAs or D-RNAs, are synthesized usingenantio-deoxyribose phosphoramidites or enantio-ribose phosphoramiditesto yield ligands of non-natural handedness which bind to the naturalconformation of the peptide corresponding to the epitope of thereceptor. This ligand of non-natural handedness will not be susceptibleto nuclease degradation by naturally occurring proteases and nucleases.

[0103] In one embodiment, the first and second binding domains areaptamers of non-natural handedness and are identified as describedabove, and the first and second binding domains are covalently coupledto provide a bivalent ligand. In one embodiment, the binding domains arecoupled via a linker.

[0104] In another embodiment, the first binding domain is an aptamer ofnon-natural handedness to a first epitope domain of a receptoridentified as described above, and the second binding domain is anaptamer of natural handedness identified by the traditional SELEX methodto a second epitope on the same 7TM G protein-coupled receptor. In yetanother embodiment, the first binding domain is an aptamer ofnon-natural handedness to a first epitope of a receptor identified asdescribed above, and the second binding domain is a non-aptamer whichbinds to a second epitope on the same 7TM G protein-coupled receptor.

[0105] 5. Linkers

[0106] In a preferred embodiment, the first and second binding domainsare covalently coupled via a linker. A linker is a molecular entity thatconnects two or more molecular entities through covalent bondsinteractions, and can allow spatial separation of the molecular entitiesin a manner that preserves the functional properties of one or more ofthe molecular entities.

[0107] In a preferred embodiment, the linker is flexible and of suitablelength such that each aptamer of the bivalent molecule is able to bindto its corresponding epitope on the same receptor simultaneously. Byflexible it is meant that the linker comprises carbon-carbon sigma bondshaving free rotation about their axes.

[0108] Suitable linkers include, but are not limited to, polymersincluding polyethylene glycol, polypropylene glycol, polyvinyl alcohol,hydrocarbons, polyacrylates and amino-, hydroxy-, thio orcarboxy-functionalized silicones; proteins; peptides; polynucleotides;saccharides including monosaccharides, oligosaccharides, cyclodextrinsand dextran; other biocompatible moieties; and combinations thereof.Such linkers are widely commercially available or obtainable viasynthetic organic methods commonly known to those skilled in the art.Methods for coupling such linkers to the ligand domains described hereininvolve standard organic synthesis and are well known to those ofordinary skill in the art. In addition, the linkers can comprise chargedfunctional groups, such as, for example, ammonium groups of carboxylategroups. The charged functional groups can provide bivalent compoundswith increased solubility in aqueous or physiological systems.

[0109] As stated above, the linkers should be of sufficient length toallow for the simultaneous binding of both aptamers to their respectiveepitopes on the same 7TM G protein-coupled receptor. Preferably, thelength of the linker is equal or greater to the distance between the twoepitopes of the receptor. This distance can be measured, or predictedtheoretically, by any method known in the art. For example, molecularmodeling can be used to determine the desired length of the linker basedon, e.g., the predicted conformation of the receptor. Molecular modelingprograms that can be used are commonly known and available in the art.

[0110] Linkers may also include lipid bilayer vesicles such asliposomes. U.S. patent application Ser. No. 08/434,465, filed May 4,1995, entitled “Nucleic Acid Ligand Complexes”, now U.S. Pat. No.6,011,020, describes complexes comprising two or more nucleic acidligands attached to the surface of the same liposome. U.S. patentapplication Ser. No. 08/739,109, filed Oct. 25, 1996, entitled “VascularEndothelial Growth Factor (VEGF) Nucleic Acid Ligand Complexes”, nowU.S. Pat. No. 5,859,228, describes complexes comprising two or moredialkyl glycerol-derivatized nucleic acid ligands attached to thesurface of the same liposome. Each of these applications is incorporatedherein by reference in their entirety. In one embodiment of the presentinvention, the bivalent binding molecules are coupled to a glycerollipid such as a diacyl glycerol and are associate with the surface of aliposome. The glycerol lipid can associate with the membrane of theliposome in such a way so that bivalent molecule is projecting out ofthe liposome.

[0111] 6. Stabilizing Nucleic Acid Ligands

[0112] In order to produce aptamers desirable for use as apharmaceutical, it is preferred that the nucleic acid ligand (1) bindsto the target in a manner capable of achieving the desired effect on thetarget; (2) be as small as possible to obtain the desired effect; (3) beas stable as possible; and (4) be a specific ligand to the chosentarget. In most situations, it is preferred that the nucleic acid ligandhave the highest possible affinity to the target.

[0113] One potential problem encountered in the therapeutic,prophylactic, and in vivo diagnostic use of nucleic acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifested.Certain chemical modifications of the nucleic acid ligand can be made toincrease the in vivo stability of the nucleic acid ligand or to enhanceor to mediate the delivery of the nucleic acid ligand. See, e.g., U.S.patent application Ser. No. 08/117,991, filed Sep. 9, 1993, entitled“High Affinity Nucleic Acid Ligands Containing Modified Nucleotides”,now abandoned and U.S. patent application Ser. No. 08/434,465, filed May4, 1995, entitled “Nucleic Acid Ligand Complexes”, now U.S. Pat. No.6,011,020, which are specifically incorporated herein by reference.Modifications of the Nucleic Acid Ligands contemplated in this inventioninclude, but are not limited to, those which provide other chemicalgroups that incorporate additional charge, polarizability,hydrophobicity, hydrogen bonding, electrostatic interaction, andfluxionality to the nucleic acid ligand bases or to the nucleic acidligand as a whole. Such modifications include, but are not limited to,2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil,backbone modifications, phosphorothioate or alkyl phosphatemodifications, methylations, unusual base-pairing combinations such asthe isobases isocytidine and isoguanidine and the like. Modificationscan also include 3′ and 5′ modifications such as capping.

[0114] Where the nucleic acid ligands are derived by the SELEX method,the modifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications yield nucleic acid ligands with both specificity for theirSELEX target and improved in vivo stability. Post-SELEX modificationsmade to 2′-OH nucleic acid ligands can result in improved in vivostability without adversely affecting the binding capacity of thenucleic acid ligand. The preferred modifications of the nucleic acidligands of the subject invention are 5′ and 3′ phosphorothioate cappingand/or 3′-3′ inverted phosphodiester linkage at the 3′ end. In onepreferred embodiment, the preferred modification of the nucleic acidligand is a 3′-3′ inverted phosphodiester linkage at the 3′ end.Additional 2′ fluoro (2′-F) and/or 2′ amino (2′-NH₂) and/or 2′ O methyl(2′-OMe) modification of some or all of the nucleotides is preferred.Included herein are nucleic acid ligands that were 2′-NH₂ modified or2′-F modified and incorporated into the SELEX process.

[0115] Other modifications are known to one of ordinary skill in theart. Such modifications may be made post-SELEX (modification ofpreviously identified unmodified ligands) or by incorporation into theSELEX process. Another example of a post-SELEX modification may beperformed as described by Schumacher et al. in PCT application No. WO96/34879, supra and in Fürste et al. in PCT application No. WO 98/08856.This method involves using an unnatural target molecule (e.g.,L-adenosine) to identify a natural D-RNA nucleic acid ligand to thattarget by the SELEX process, followed by synthesis of the mirror-imageof the ligand, i.e., the unnatural L-RNA, which recognizes the naturalconformation of the intended target. These unnatural L-RNA nucleic acidligands are therefore stable in that they are not degraded by nucleasespresent in human serum, which only recognize natural D-RNA.

[0116] 7. Uses of Bivalent Binding Molecules

[0117] The bivalent binding molecules identified by the methodsdescribed herein are useful for both therapeutic and diagnosticpurposes. For example, the bivalent binding molecules of the presentinvention are useful for activating 7TM G protein-coupled receptorsspecific for that bivalent molecule so as to elicit an appropriatesecond messenger response, i.e., to initiate a desired physiologicalresponse. It is believed that in embodiments wherein the bivalentbinding molecules comprise two ligand domains to two different epitopeson the same 7TM G-protein receptor, where each ligand domain is anagonist of its corresponding epitope, that a physiological response willoccur with synergistic efficacy greater that that of the individualligands.

[0118] The bivalent binding molecules of the invention are thus usefulfor treating a disease or condition mediated by a 7TM G protein-coupledreceptor. A disease or condition is said to be mediated by a receptor ifthe symptoms associated with it are caused by or exacerbated by bindingof the receptor. It is well within the ability of skilled artisans todetermine whether a disease or condition is mediated by a particularreceptor. Thus for example, the bivalent binding molecules of theinvention may act as antagonists in which they bind to the receptor butdo not transmit a signal across the cell membrane in which the receptorresides. The bivalent binding molecules can compete with the naturalligand of the receptor and thereby reduce or prevent interaction betweenthe receptor and the natural ligand. The bivalent binding molecules mayalso modulate the activity of the receptor by altering the events thatoccur after the receptor is bound. For example, the bivalent moleculecan alter the interaction between the receptor and the G protein withwhich it naturally interacts, or alter phosphorylation sites present inthe intracellular domains of the receptor.

[0119] The bivalent binding molecules of the invention may also act asagonists wherein ligand domains of the bivalent molecule bind to theirrespective epitopes on the same receptor and transmit a signal acrossthe cell membrane.

[0120] Therapeutic uses include the treatment or prevention of diseasesor medical conditions mediated by 7TM G protein-coupled receptors inhuman patients, comprising administering an effective amount of abivalent compound of the present invention. Diseases mediated by 7TM Gprotein-coupled receptors include inflammatory diseases such as asthma,chronic obstructive pulmonary disease, cystic fibrosis, sinusitis,rhinitis, atherosclerosis, glomerulonephritis, multiple sclerosis, andinflammatory bowel disease. The disease may also be a neurologicaldisease, such as Alzheimer's disease. Therapeutic uses also include theregulation of systolic and/or diastolic blood pressure.

[0121] Diagnostic utilization may include both in vivo or in vitrodiagnostic applications. The SELEX method generally, and the specificadaptations of the SELEX method taught and claimed herein specifically,are particularly suited for diagnostic applications. The SELEX methodidentifies nucleic acid ligands that are able to bind targets with highaffinity and with surprising specificity. These characteristics are, ofcourse, the desired properties one skilled in the art would seek in adiagnostic ligand.

[0122] In embodiments of the invention wherein the bivalent bindingmolecules of the present invention comprise aptamers, the bivalentbinding molecules may be routinely adapted for diagnostic purposesaccording to any number of techniques employed by those skilled in theart. Diagnostic agents need only be able to allow the user to identifythe presence of a given target at a particular locale or concentration.Simply the ability to form binding pairs with the target may besufficient to trigger a positive signal for diagnostic purposes. Thoseskilled in the art would also be able to adapt any nucleic acid ligandby procedures known in the art to incorporate a labeling tag in order totrack the presence of such ligand. Such a tag could be used in a numberof diagnostic procedures. The bivalent binding molecules describedherein may specifically be used for identification of 7TM Gprotein-coupled receptors.

[0123] 8. Therapeutic Compositions of Bivalent Binding Molecules

[0124] Various delivery systems are known in the art and can be used toadminister the bivalent compounds of the invention, e.g., aqueoussolution, encapsulation in liposomes, microparticles, microcapsules.

[0125] Therapeutic compositions of the nucleic acid ligands may beadministered parenterally by injection, although other effectiveadministration forms, such as intra-articular injection, inhalant mists,orally active formulations, transdermal iontophoresis or suppositoriesare also envisioned. One preferred carrier is physiological salinesolution, but it is contemplated that other pharmaceutically acceptablecarriers may also be used. In one preferred embodiment, it is envisionedthat the carrier and the nucleic acid ligand constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

[0126] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or dehydrated or lyophilized powder. Such formulations may be storedeither in a ready to use form or requiring reconstitution immediatelyprior to administration. The manner of administering formulationscontaining nucleic acid ligands for systemic delivery may be viasubcutaneous, intramuscular, intravenous, intranasal or vaginal orrectal suppository.

[0127] 9. Effective Dose

[0128] The amount of the bivalent compound which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, which can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness or advancement of thedisease or condition, and should be decided according to thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curved derived from in vitro or animalmodel test systems. For example, an effective amount of a bivalentcompound of the invention is readily determined by administering gradeddoses of a bivalent compound of the invention and observing the desiredeffect.

[0129] The following examples are meant to illustrate the method of theinvention and are not intended to limit the scope, utility, orapplicability of this invention.

EXAMPLE 1 Identification of a Bivalent Binding Molecule to NK1R

[0130] The biological actions of substance P, a neurokinin, is mediatedby a neurokinin receptor known as NK1R. This receptor is a member of the7TM G protein-coupled receptor superfamily. Studies have shown that theextracellular domains of the NK1R comprise the ligand binding sites(Fong, T. M. et al. Journal of Biological Chem. 267:25664-25667 (1992)).This example describes the identification of a bivalent molecule to NK1Rhaving affinity for epitopes in extracellular loop 1 and extracellularloop 2.

[0131] a) Synthesis of Peptide Target 1 (Epitope 1).

[0132] Extracellular loop 1 (ECL1) of NK1R is comprised of the 13 aminoacid sequence: N-His-Asn-Glu-Trp-Tyr-Tyr-Gly-Leu-Phe-Tyr-Cys-Lys-Phe-C(Fong, et al.) (SEQ. ID NO. 1). ECL1 is synthesized by standard peptidesynthesis methods known in the art with an additional Cys at the carboxyterminus (ECL1-Cys) to facilitate coupling to a support. ECL1-Cys iscovalently coupled (via a disulfide bond) to thiopropyl-activatedSEPHAROSE™ 6B matrix through an interaction of the Cys-thiol group ofpeptides with hydroxypropyl-2-pyridyldisulfide ligands of the matrix asdescribed in U.S. Pat. No. 5,648,214, (Nieuwlandt, et al. supra).

[0133] b) Synthesis of Peptide Target 1 (Epitope 2).

[0134] Extracellular loop 2 (ECL2) of NK1R is comprised of the 26 aminoacid sequence:Thr-Thr-Glu-Thr-Met-Pro-Ser-Arg-Val-Val-Cys-Met-Ile-Glu-Trp-Pro-Glu-His-Pro-Asn-Lys-Ile-Tyr-Glu-Lys-Val(Fong, et al.). ECL2 is synthesized by standard peptide synthesismethods known in the art with an additional Cys at the carboxy terminus(ECL2-Cys) to facilitate coupling to a support. ECL2-Cys is covalentlycoupled (via a disulfide bond) to thiopropyl-activated Sepharose 6Bthrough an interaction of the Cys-thiol group of peptides withhydroxypropyl-2-pyridyldisulfide ligands of the matrix as described inU.S. Pat. No. 5,648,214, (Nieuwlandt, et al. supra).

[0135] c) Random Sequence RNA Pool.

[0136] Template DNA for the initial random sequence RNA population isgenerated from a synthetic random sequence ssDNA pool. The random regionis generated by utilizing a mixture of the four unmodified nucleotides(the molar ratios of which are adjusted to yield a 1:1:1:1 ratio ofincorporated nucleotides) during oligonucleotide synthesis. The ssDNAscontain 30 nucleotides of contiguous random sequence flanked by defined5′ and 3′ ends that permit primer hybridization. Double-stranded DNA(dsDNA) molecules, synthesized initially by Klenow enzyme, andsubsequently (following cycles of selection) by Taq DNA polymerase, havea T7 RNA polymerase promoter at the 5′ end. In vitro transcription ofthe dsDNA template yields the initial pool of uniformly[a-³²P]GTP-labeled random sequence RNAs.

[0137] d) Identification of an Aptamer Library 1 to Peptide Target 1.

[0138] Uniformly ³²P-labeled RNAs are suspended in an appropriatebinding buffer. The suspension is applied to the ECL1-Cys SEPHAROSE™column, followed by binding buffer wash volumes. Peptide-bound RNAs arethen recovered with washes of binding buffer containing dithiothreitol(DTT). DTT reduces the linker disulfide bond resulting in the release ofpeptide from the matrix. The DTT eluate is extracted with phenol and theRNAs are recovered by ethanol precipitation with yeast tRNA as carrier.Reverse transcription, PCR amplification and T7 RNA polymerasetranscription are performed essentially as described in Tuerk and Gold(Science 249:505-510 (1990)). Transcription of PCR products yield theRNA pool for the next cycle of selection and amplification. Cycles ofselection and amplification are performed as many times as desired toidentify high affinity RNA aptamers to epitope 1.

[0139] e) Identification of an Aptamer Library 2 to Peptide Target 2.

[0140] The protocol described in paragraph d) is followed using theECL-2-Cys SEPHAROSE™ column to obtain high affinity RNA aptamers toepitope 2.

[0141] f) Overlap-extension (OE) Reaction.

[0142] Prior to the formation of bivalent binding molecules,double-stranded DNA is generated by PCR from each library in such a waythat the 3′ fixed sequence of library 1 is identical to the 5′ fixedsequence of library 2. Specifically, the 5′ primer normally used toamplify library 2 is replaced by a primer that lacks sequencescontaining the transcriptional promoter. Amplified material from eachlibrary is gel purified, and the two libraries are mixed in an OEreaction.

[0143] In OE, the DNA is denatured at high temperature and allowed toanneal at 55° C. prior to extension at 72° C. with Taq DNA polymerase.Then two primers, corresponding to the 5′ fixed region of library 1 andthe 3′ fixed region of library 2 are added and PCR performed to generatedouble stranded DNA to be used as template for further cycles ofselection/amplification.

[0144] g) Enrichment of the Bivalent Binding Molecule Library

[0145] Cycles of the SELEX process are carried out using the bivalentbinding molecule library as described in paragraph d) using the peptide1 affinity column. The bivalent binding molecules which bind to thecolumn are collected and used as the pool in cycles of the SELEX processusing the peptide affinity column 2. The bivalent binding molecules thatbind to peptide affinity column 2 are collected and screened for theirability to bind NK1R.

[0146] h) Selection for NK1R Affinity

[0147] The affinity of the enriched bivalent molecule library for NK1Rexpressed in cells is carried out using the SELEX procedure described inU.S. Pat. No. 5,750,342, filed May 3, 1995, entitled “Nucleic AcidLigands of Tissue Target”, followed by a negative selection using anidentical cell line which does not express NK1R. The bivalent bindingmolecules isolated bind both epitopes 1 and 2 of NK1R. TABLE 1^(a)Cloned and expressed seven transmembrane receptors Receptor SpeciesSystem Adenosine A₁ rat A9-L + CHO human CHO canine CHO A_(2b) ratXenopus oocytes human CHO-K1 A₃ sheep COS-1/CHO K1 A₃ human CHOAdrenergic α₁ hamster COS-7 α₁ bovine COS-7 α₂ human COS-7 CHO mouseCOS-7 Xenopus oocytes fish COS-7 α_(2C) mouse rat CHO/COS-7 α_(2A) humanCOS-7 human COS 1 α_(2B), α_(2C), α_(2D) rat NIH 3T3 α_(2b) rat COShuman Ltk cells α_(2D) rat COS 1 α_(2A) human Xenopus oocytes porcineCOS-M6 α_(2B) rat COS α_(1D) human SK-N-MC β₁ rat L cells human Xenopusoocytes mouse COS-7/L-cells sf9 β₁/β₂ human CHO β₂ mouse Y-1 humanEscherichia coli β₃ mouse CHO rat CHO human CHO cells Calcium bovineXenopus oocytes Dopamine D₁ human rat COS-7 human COS-7 rhesus C6 cellsD_(1B) rat COS-7 D₂ human COS-7 mouse COS-7 rat COS-7 CHO-6, DUK 25 ratmouse fibroblasts D₂/D₃ human CHO D₂/D₃ rat LZR1, Ltk 59 D₃ rat CHO D₄human COS-7 D₅ human COS-7 Histamine H₁ bovine COS-7 H₂ rat CHO canineL-cells human Colo-320 Muscarininc muscarininc porcine Xenopus oocytesmuscarininc porcine CHO muscarininc drosoph. Y-1 cells m1 mouse Y-1,L-cells human CHO-K1 m1, m2 human HEK m2 human CHO-K1 m3 human CHO-K1CHO m4 chicken Y1/CHO human CHO-K1 m5 human/rat COS-7 CHO-K1 Opioid κmouse PC-12 COS-1 human COS-1 rat COS-7/Xenopus oocytes COS-7 δ mouseCHO-DGH4 human CHO COS μ rat COS-7 COS-7 COS-7 δ human COS-7 mouse COS-1μ human COS-7 Peptides Angiotensisn type 2 human COS-7 mouse COS-7 ratCOS-7 Bradykinin B₂ rat Xenopus oocytes human COS-7 Calcitonin human ratpig COS Cholecystokin A human COS rat Xenopus oocytes B human COS-7Choriogonadotropin porcine COS-7 Corticotropin releasing factor ratCOS-7 Endothelin_(B) human COS-7 Gastrin canine COS-7 Glucagon rat BHKGonadotropin releasing hormone human COS-7 Growth hormone releasinghormone rat HEK 293 Lutropin/luteinizing hormone rat HEK 292 mouse Lcells Neuropeptide Y rat 293 Neurotensin_(A) human BaculovirusParathyroid hormone opossum COS-7 rat COS Secretin rat COS human HEK 293Somatostatin mouse/human CHO R1 rat COS-7 human CHO R2 mouse CHO R3 ratCOS human COS-1 mouse CHO R4 human COS-7 human COS-1 mouse COS-1DM R4,R5 human CHO-K1/COS-1 R5 human CHO-K1 human COS-7 Substance P murineXenopus oocytes rat COS human COS-7 TSH human COS-7 Thyrotropin canineCOS rat CHO-K1 VIP rat COP human COS-6 5-HT 1 rat HEK 293 1A rat Ltkhuman monkey kidney NIH 3T3 1B rat Y-1 human sf9 HeLa mouse NIH 3T3 1Chuman Xenopus oocytes mouse Xenopus oocytes 1D canine COS-7 human CHO-K11E human murine L cells 2 rat (mammalian) 2B human AV12-664 3 mouseCOS-1/Xenopus oocytes 5A, 5B mouse COS-7 5A human Cos M6 (S12) human Ltk7 rat COS-7, HEK 293 GP2-7 guinea pig CHO-K1

[0148]

1 2 1 13 PRT Homo sapiens 1 His Asn Glu Trp Tyr Tyr Gly Leu Phe Tyr CysLys Phe 1 5 10 2 26 PRT Homo sapiens 2 Thr Thr Glu Thr Met Pro Ser ArgVal Val Cys Met Ile Glu Trp Pro 1 5 10 15 Glu His Pro Asn Lys Ile TyrGlu Lys Val 20 25

1. A bivalent binding molecule comprising two or more binding domains totwo or more epitopes of the same 7 transmembrane G protein-coupledreceptor, wherein the binding domains are coupled to each other.
 2. Thebivalent binding molecule of claim 1 wherein at least one binding domainis an aptamer.
 3. The bivalent binding molecule of claim 2, wherein saidaptamer is a SELEX-derived aptamer.
 4. The bivalent binding molecule ofclaim 1, wherein all binding domains are aptamers.
 5. The bivalentbinding molecule of claim 1, wherein one binding domain is an aptamerand the other binding domains are non-aptamer binding domains.
 6. Thebivalent binding molecule of claim 1 wherein the binding domains arecoupled to each other via a linker.
 7. The bivalent binding molecule ofclaim 6 wherein said linker is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, polyvinyl alcohol,hydrocarbons, polyacrylates and amino-, hydroxy-, thio orcarboxy-functionalized silicones, proteins, peptides, polynucleotides,monosaccharides, oligosaccharides, cyclodextrins, dextran and liposomes.8. The bivalent binding molecule of claim 2 wherein the aptamer bindingdomain is coupled at the 3′ end to another binding domain.
 9. Thebivalent binding molecule of claim 2 wherein the aptamer binding domainis coupled at the 5′ end to another binding domain.
 10. The bivalentbinding molecule of claim 2 wherein said aptamer is modified at the 2′,5 or 8 position.
 11. The bivalent binding molecule of claim 1, whereinsaid 7 transmembrane G protein-coupled receptor is selected from thereceptors in Table
 1. 12. A bivalent binding molecule to a 7transmembrane G protein-coupled receptor, wherein said bivalent bindingmolecule comprises a first and second aptamer to a first and secondepitope of the same 7 transmembrane G protein-coupled receptor, saidbivalent binding molecule identified by the method comprising: a)identifying said first aptamer to said first epitope by the methodcomprising: i) preparing a first candidate mixture of nucleic acids; ii)contacting said first candidate mixture of nucleic acid with said firstepitope, wherein nucleic acids having an increased affinity to saidfirst epitope may be partitioned from the remainder of the firstcandidate mixture; iii) partitioning said increased affinity nucleicacids from the remainder of the first candidate mixture; and iv)amplifying said increased affinity nucleic acids, whereby said firstaptamer to said first epitope may be identified; b) identifying saidsecond aptamer to said second epitope by the method comprising: i)preparing a second candidate mixture of nucleic acids; ii) contactingsaid second candidate mixture of nucleic acid with said second epitope,wherein nucleic acids having an increased affinity to said secondepitope may be partitioned from the remainder of the first candidatemixture; p2 iii) partitioning said increased affinity nucleic acids fromthe remainder of the first candidate mixture; and iv) amplifying saidincreased affinity nucleic acids, whereby said second aptamer to saidsecond epitope may be identified; and c) coupling said first aptamer tosaid second aptamer, whereby said bivalent binding molecule may beidentified.
 13. A bivalent binding molecule to a 7 transmembrane Gprotein-coupled receptor, wherein said bivalent binding moleculecomprises a first and second aptamer to a first and second epitope ofsaid same 7TM G protein-coupled receptor, wherein said bivalent bindingmolecule is identified by a method comprising: a) preparing a bivalentbinding molecule library generated according to a method comprising: i)generating a first library of aptamers selected through the SELEXprocedure for binding to said first epitope of said 7 transmembrane Gprotein-coupled receptor said aptamers having a 3′ fixed sequence, andproducing the double-stranded form of said first library of aptamers;ii) generating a second library of aptamers selected through the SELEXprocedure for binding to said second epitope of said 7 transmembrane Gprotein-coupled receptor, said aptamers having a 5′ fixed sequenceidentical to the 3′ fixed sequence of the aptamers of said firstlibrary, and producing the double-stranded form of said second libraryof aptamers; iii) mixing said first and second libraries underconditions which promote interlibrary annealing; iv) forming bivalentbinding molecules by enzymatically extending the recessed 3′ ends whilecopying the 5′ extensions of each annealed pair, to yield adouble-stranded bivalent binding molecule library; v) amplifying saiddouble-stranded bivalent binding molecule library; b) contacting saidbivalent binding molecule library with said 7 transmembrane Gprotein-coupled receptor, wherein bivalent binding molecules having anincreased affinity to said first and second epitopes of said 7transmembrane G protein-coupled receptor may be partitioned from theremainder of the bivalent binding molecule library; c) partitioning saidincreased affinity bivalent binding molecules from the remainder of saidbivalent binding molecule library; d) amplifying said increased affinitybivalent binding molecules to yield a mixture increased affinitybivalent binding molecules having increased affinity to said first andsecond epitopes, whereby bivalent binding molecules to a 7 transmembraneG protein coupled receptor having affinity to a first and second epitopemay be identified.
 14. A bivalent binding molecule to a 7 transmembraneG protein-coupled receptor, wherein said bivalent binding moleculecomprises an aptamer to a first epitope coupled to a non-aptamer bindingdomain which binds to a second epitope of the same receptor, wherein thebivalent binding molecule is identified according to a methodcomprising: a) preparing a blended candidate mixture of bivalent bindingmolecules comprising a candidate mixture of nucleic acid sequencescoupled to a non-aptamer binding domain which binds to said secondepitope of the receptor; b) contacting said 7 transmembrane Gprotein-coupled receptor with said blended candidate mixture of bivalentbinding molecules, wherein bivalent binding molecules having anincreased affinity to the 7 transmembrane G protein-coupled receptorrelative to the blended candidate mixture may be partitioned from theremainder of the candidate mixture; c) partitioning the increasedaffinity bivalent binding molecules from the remainder of the blendedcandidate mixture; and d) amplifying the increased affinity bivalentbinding molecules to yield an enriched mixture of bivalent bindingmolecules, whereby bivalent binding molecules to a 7 transmembrane Gprotein-coupled receptor may be identified.
 15. A bivalent bindingmolecule to a 7 transmembrane G protein-coupled receptor, wherein saidbivalent binding molecule comprises an aptamer to a first epitope on afirst extracellular domain of said receptor coupled to a non-aptamerbinding domain which binds to a second epitope on a second extracellulardomain of the same 7TM G protein-coupled receptor, wherein said bivalentbinding molecule is identified according to a method comprising: a)identifying an aptamer to said first epitope of said 7 transmembrane Gprotein-coupled receptor by the method comprising: i) preparing acandidate mixture of nucleic acids; ii) contacting said candidatemixture with said first epitope, wherein nucleic acids having anincreased affinity to said first epitope relative to the candidatemixture may be partitioned from the remainder of the candidate mixture;iii) partitioning the increased affinity nucleic acids from theremainder of the candidate mixture; and iv) amplifying the increasedaffinity nucleic acids to yield an enriched mixture of nucleic acids,whereby an aptamer to said first epitope of said 7 transmembrane Gprotein-coupled receptor may be identified; and b) coupling said aptamerto a non-aptamer binding domain which binds to said second epitope ofsaid 7 transmembrane G protein coupled receptor to yield a bivalentbinding molecule.
 16. A bivalent binding molecule to a 7 transmembrane Gprotein-coupled receptor, wherein said bivalent binding moleculecomprises an aptamer coupled to a second binding domain, said aptamerbeing an aptamer of non-natural handedness having binding affinity tothe natural configuration of a first epitope, wherein said bivalentbinding molecule is identified according to a method comprising: a)identifying said aptamer of non-natural handedness by the methodcomprising: i) synthesizing a peptide enantiomer of the naturalconfiguration of an amino acid sequence corresponding to a first epitopeof said 7 transmembrane G protein-coupled receptor; ii) contacting saidpeptide enantiomer with a candidate mixture of nucleic acids of naturalhandedness, wherein nucleic acids of natural handedness having anincreased affinity to the peptide enantiomer relative to the candidatemixture may be partitioned from the remainder of the candidate mixture;iii) partitioning the increased affinity nucleic acids of naturalhandedness from the remainder of the candidate mixture; iv) amplifyingthe increased affinity nucleic acids of natural handedness; v)identifying the sequences of said increased affinity nucleic acids ofnatural handedness; and vi) synthesizing the enantiomers of saidincreased affinity nucleic acids of natural handedness to yield amixture of increase affinity nucleic acids of non-natural handedness,whereby an aptamer of non-natural handedness to the naturalconfiguration of said first epitope may be identified; and b) covalentlycoupling said aptamer of non-natural handedness to a second bindingdomain of said second epitope of said 7 transmembrane G protein-coupledreceptor, whereby a bivalent binding molecule of said 7 transmembrane Gprotein-coupled receptor may be identified.